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. 1986 Nov;54(2):537–542. doi: 10.1128/iai.54.2.537-542.1986

Cloning and expression of the Bacillus anthracis protective antigen gene in Bacillus subtilis.

B E Ivins, S L Welkos
PMCID: PMC260194  PMID: 3021632

Abstract

The gene encoding the protective antigen (PA) moiety of the tripartite exotoxin of Bacillus anthracis was cloned from the recombinant plasmid pSE36 into Bacillus subtilis 1S53 by using the plasmid vector pUB110. Two clones, designated PA1 and PA2, were identified which produced PA in liquid cultures at levels of 20.5 to 41.9 micrograms/ml. This PA was identical to B. anthracis Sterne PA with respect to migration on sodium dodecyl sulfate-polyacrylamide gels and to Western blot antigenic reactivity. Addition of lethal factor or edema factor to PA1 and PA2 supernatants generated biologically active anthrax lethal toxin or edema-producing toxin, respectively. The recombinant plasmid in PA1 (pPA101) was 7.8 kilobases, whereas the PA2 strain plasmid (pPA102) was 6.1 kilobases. Both plasmids had deletions extending into the insert sequence but not into the DNA encoding the PA protein. Immunization with the live recombinant strains protected guinea pigs from lethal challenge with virulent B. anthracis spores, and the immunization partially or completely protected rats from intravenous challenge with anthrax lethal toxin.

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  1. BEALL F. A., TAYLOR M. J., THORNE C. B. Rapid lethal effect in rats of a third component found upon fractionating the toxin of Bacillus anthracis. J Bacteriol. 1962 Jun;83:1274–1280. doi: 10.1128/jb.83.6.1274-1280.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chang S., Cohen S. N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. doi: 10.1007/BF00267940. [DOI] [PubMed] [Google Scholar]
  3. DEARMON I. A., Jr, KLEIN F., LINCOLN R. E., MAHLANDT B. G., FERNELIUS A. L. Immunological studies of anthrax. I. An index to determine quantitative immunization. J Immunol. 1961 Sep;87:233–239. [PubMed] [Google Scholar]
  4. DULBECCO R., VOGT M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 1954 Feb;99(2):167–182. doi: 10.1084/jem.99.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ezzell J. W., Ivins B. E., Leppla S. H. Immunoelectrophoretic analysis, toxicity, and kinetics of in vitro production of the protective antigen and lethal factor components of Bacillus anthracis toxin. Infect Immun. 1984 Sep;45(3):761–767. doi: 10.1128/iai.45.3.761-767.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gryczan T. J., Contente S., Dubnau D. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J Bacteriol. 1978 Apr;134(1):318–329. doi: 10.1128/jb.134.1.318-329.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gryczan T. J., Dubnau D. Construction and properties of chimeric plasmids in Bacillus subtilis. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1428–1432. doi: 10.1073/pnas.75.3.1428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Guerry P., LeBlanc D. J., Falkow S. General method for the isolation of plasmid deoxyribonucleic acid. J Bacteriol. 1973 Nov;116(2):1064–1066. doi: 10.1128/jb.116.2.1064-1066.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. HAINES B. W., KLEIN F., LINCOLN R. E. QUANTITATIVE ASSAY FOR CRUDE ANTHRAX TOXINS. J Bacteriol. 1965 Jan;89:74–83. doi: 10.1128/jb.89.1.74-83.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. HARRIS-SMITH P. W., SMITH H., KEPPIE J. Production in vitro of the toxin of Bacillus anthracis previously recognized in vivo. J Gen Microbiol. 1958 Aug;19(1):91–103. doi: 10.1099/00221287-19-1-91. [DOI] [PubMed] [Google Scholar]
  11. Hahn J., Dubnau D. Analysis of plasmid deletional instability in Bacillus subtilis. J Bacteriol. 1985 Jun;162(3):1014–1023. doi: 10.1128/jb.162.3.1014-1023.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ivins B. E., Ezzell J. W., Jr, Jemski J., Hedlund K. W., Ristroph J. D., Leppla S. H. Immunization studies with attenuated strains of Bacillus anthracis. Infect Immun. 1986 May;52(2):454–458. doi: 10.1128/iai.52.2.454-458.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jalanko A., Palva I., Söderlund Restriction maps of plasmids pUB110 and pBD9. Gene. 1981 Sep;14(4):325–328. doi: 10.1016/0378-1119(81)90165-7. [DOI] [PubMed] [Google Scholar]
  14. Kawamura F., Doi R. H. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J Bacteriol. 1984 Oct;160(1):442–444. doi: 10.1128/jb.160.1.442-444.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Leppla S. H. Bacillus anthracis calmodulin-dependent adenylate cyclase: chemical and enzymatic properties and interactions with eucaryotic cells. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;17:189–198. [PubMed] [Google Scholar]
  17. Little S. F., Knudson G. B. Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig. Infect Immun. 1986 May;52(2):509–512. doi: 10.1128/iai.52.2.509-512.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McDonald K. O., Burke W. F., Jr Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis. J Bacteriol. 1982 Jan;149(1):391–394. doi: 10.1128/jb.149.1.391-394.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Millet J. Characterization of proteinases excreted by Bacillus subtilis Marburg strain during sporulation. J Appl Bacteriol. 1970 Mar;33(1):207–219. doi: 10.1111/j.1365-2672.1970.tb05245.x. [DOI] [PubMed] [Google Scholar]
  20. PUZISS M., HOWARD M. B. Studies on immunity in anthrax. XI. Control of cellular permeability by bicarbonate ion in relation to protective antigen elaboration. J Bacteriol. 1963 Jan;85:237–243. doi: 10.1002/path.1700850123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. PUZISS M., MANNING L. C., LYNCH J. W., BARCLAYE, ABELOW I., WRIGHT G. G. Large-scale production of protective antigen of Bacillus anthracis in anaerobic cultures. Appl Microbiol. 1963 Jul;11:330–334. doi: 10.1128/am.11.4.330-334.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. PUZISS M., WRIGHT G. G. Studies on immunity in anthrax. IV. Factors influencing elaboration of the protective antigen of bacillus antracis in chemically defined media. J Bacteriol. 1954 Oct;68(4):474–482. doi: 10.1128/jb.68.4.474-482.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ristroph J. D., Ivins B. E. Elaboration of Bacillus anthracis antigens in a new, defined culture medium. Infect Immun. 1983 Jan;39(1):483–486. doi: 10.1128/iai.39.1.483-486.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. STANLEY J. L., SMITH H. Purification of factor I and recognition of a third factor of the anthrax toxin. J Gen Microbiol. 1961 Sep;26:49–63. doi: 10.1099/00221287-26-1-49. [DOI] [PubMed] [Google Scholar]
  25. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tsao S. G., Brunk C. F., Pearlman R. E. Hybridization of nucleic acids directly in agarose gels. Anal Biochem. 1983 Jun;131(2):365–372. doi: 10.1016/0003-2697(83)90185-9. [DOI] [PubMed] [Google Scholar]
  27. Vodkin M. H., Leppla S. H. Cloning of the protective antigen gene of Bacillus anthracis. Cell. 1983 Sep;34(2):693–697. doi: 10.1016/0092-8674(83)90402-6. [DOI] [PubMed] [Google Scholar]
  28. WRIGHT G. G., HEDBERG M. A., FEINBERG R. J. Studies on immunity in anthrax. II. In vitro elaboration of protective antigen by non-proteolytic mutants of Bacillus anthracis. J Exp Med. 1951 Jun;93(6):523–527. doi: 10.1084/jem.93.6.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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